The present invention relates generally to illumination apparatuses used for purposes such as fluorescence microscopy, and specifically to an illumination apparatus comprising a multiple wavelength light emitting diode (LED) array and its accompanying optical elements.
Fluorescence microscopy is popularly used in numerous bio/medical applications since it enables users to label and observe specific structures or molecules. Briefly, fluorescence is a chemical process where when light of a specific wavelength is shined upon a fluorescent molecule, electrons are excited to a high energy state in a process known as excitation. These electrons remain briefly in this high energy state, for roughly a nanosecond, before dropping back to a low energy state and emitting light of a lower wavelength. This process is referred to as fluorescent emission, or alternatively as fluorescence.
In a typical fluorescence microscopy application, one or more types of fluorescent materials or molecules (sometimes referred to as fluorescent dyes) are used, along with an illuminator apparatus that provides the exciting wavelength, or wavelengths. Different fluorescent molecules can be selected to have visually different emission spectra. Since different fluorescent molecules typically have different excitation wavelengths, they can be selectively excited so long as the bandwidth of the excitation light for one fluorescent molecule does not overlap the excitation wavelengths of other fluorescent molecules that are being used in the same experiment. Therefore the excitation light should ideally have a narrow bandwidth and have its peak output at the excitation wavelength of the molecule in question. Furthermore, fluorescence is a probabilistic event with low signal levels so an intense light is typically used to increase the chances of the process occurring. Most fluorescence microscopy applications also benefit from having a uniformly intense illuminated field of view or area, ideally such that the size and shape of the illuminated area can be modified. Simultaneously achieving all these criteria has been difficult but is necessary for current and future applications that require increasing levels of illumination control and consistency.
Traditional prior art fluorescence microscopy illuminators have relied on metal halide arc lamp bulbs such as Xenon or Mercury bulbs, as light sources. The broad wavelength spectrum produced by these lamps when combined with specific color or band pass filters allows for the selection of different illumination wavelengths. This wavelength selection and light shaping process, however, is highly energy inefficient since in selecting only a relatively small portion of wavelength spectrum produced by the Xenon or Mercury bulb, the vast majority of the light outputted from the lamp is unused. These wavelength selection or band pass filters are costly, especially when placed on a mechanical rotating wheel in typical multiple-wavelength applications.
In this type of implementation using metal halide arc lamp bulbs, the speed with which different wavelengths can be selected is limited by the mechanical motion of moving various filters into place. In addition to the sluggishness and unreliability of filter wheels, as well as energy coupling inefficiency, metal halide arc lamps are also hampered by the limited lifetime of the bulb, typically ˜2000 hours. The intensity of the light output declines with bulb use and once exhausted, the user has to undergo a complicated and expensive process of replacing the bulb and subsequently realigning the optics without any guarantee that the illuminator will perform as before. These disadvantages make acquiring consistent results difficult and inconvenient for users who must deal with the variable output of the bulbs, and who must either be trained in optical alignment or call upon professionals when a bulb needs to be replaced.
In recent years, several prior art multiple wavelength illuminators have been developed using different colored LEDs as light sources, that overcome numerous limitations of metal halide arc lamps. Not only do they last longer, with the lifetime of an LED chip being typically rated at well over 10,000 hours, but in addition the power output varies negligibly over that period. Furthermore, the bandwidth of the spectral output of an LED chip is typically narrow (<30 nm) which can eliminate the need for additional band pass filters and is ideal for fluorescence applications. The intensity of the output light can be quickly and accurately controlled electronically by varying the current through the LED chip(s), whereas in metal halide illuminators, the output intensity of the bulb is constant and apertures or neutral density filters are used to attenuate the light entering the microscopy.
Prior art LED illuminators for fluorescence microscopy have thus far used up to 5 separate LED modules, each containing one up to a few chips, for each wavelength. Since the LED chips in these modules have their own individual packaging, the modules are large so that light beams emitted from the modules will need to be combined using optical elements. Although such prior art LED illuminators allow the user the flexibility to swap out modules for new modules with different wavelengths, the additional elements such as lenses, mirrors and heat sinks required for each separate color add complexity, bulk and cost. Furthermore, the long optical paths required to combine the beams from multiple LED chips or modules that are spatially separated, make it difficult to collect and shape already highly divergent light coming from the LED chips. These practical issues have limited the application of such illuminators in fluorescence microscopy, which in general requires light that is both intense and spatially uniform.